Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 2012-07-16 to 2012-08-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodoligcal deficiencies, which do not affect the quality of the relevant results. the study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions on a similar substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Alkenes hydroformylated, sulfosuccinates, sodium salt
- Physical state: solid (waxy), yellowish
- Lot no. / Batch no.: Rif. 7018
- Purity : 100 %; active ingredient: 78 %; dose calculation was no adjusted to purity
- Expiration date of the lot/batch: 2013-05-31
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: In the freezer at ≤ - 18°C

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation

Migrated to IUCLID6: sodium azide, NaN3 used with Strains TA 1535, TA 100 @ 10 µg/plate
Negative controls:
yes
Solvent controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD used with Strains: TA 1537, TA 98 @ 10 µg/plate in TA 98, 50 µg/plate in TA 1537
Remarks:
Without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation

Migrated to IUCLID6: methyl methane sulfonate, MMS used with Strains: WP2 uvrA @ 3 µL/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA used with Strains:TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA @ 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),10 µg/plate (WP2 uvrA)
Remarks:
With metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/mL (lot no. R 260412) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.



Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1535 at 2500 and 5000 µg/plate in the absence of metabolic activation. No toxic effects were observed in the remaining strain with and without metabolic activation.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Study Name: 1491203

Study Code: Harlan CCR 1491203

Experiment: 1491203 VV Plate

Date Plated: 16/07/2012

Assay Conditions:

Date Counted: 19/07/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

10 ± 4

11 ± 4

24 ± 2

176 ± 19

51 ± 1

Untreated

 

 

17 ± 4

9 ± 5

28 ± 3

161 ± 13

39 ± 7

Alkenes,

3 µg

 

12 ± 2

9 ± 4

27 ± 5

167 ± 27

47 ± 7

hydroformylated

10 µg

 

9 ± 1

9 ± 5

29 ± 4

178 ± 14

45 ± 4

sulfosuccinates,

33 µg

 

13 ± 4

9 ± 3

22 ± 5

176 ± 1

46 ± 4

sodium salt

100 µg

 

9 ± 1

10 ± 3

27 ± 9

185 ± 6

55 ± 8

 

333 µg

 

10 ± 2

13 ± 4

23 ± 5

164 ± 11

45 ± 7

 

1000 µg

 

12 ± 6

9 ± 3

25 ± 4

162 ± 16

56 ± 16

 

2500 µg

 

9 ± 1P

8 ± 2P

30 ± 8P

163 ± 26P

51 ± 13P

 

5000 µg

 

16 ± 5P

12 ± 3P

30 ± 5P

173 ± 9P

55 ± 6P

NaN3

10 µg

 

1835 ± 56

 

 

1967 ± 122

 

4-NOPD

10 µg

 

 

 

297 ± 19

 

 

4-NOPD

50 µg

 

 

67 ± 7

 

 

 

MMS

3.0 µL

 

 

 

 

 

997 ± 58

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

19 ± 8

22 ± 4

40 ± 6

199 ± 17

58 ± 6

Untreated

 

 

19 ± 9

18 ± 1

40 ± 9

171 ± 7

53 ± 9

Alkenes,

3 µg

 

22 ± 4

22 ± 4

41 ± 3

177 ± 6

52 ± 5

hydroformylated

10 µg

 

21 ± 6

25 ± 3

35 ± 6

196 ± 6

44 ± 1

sulfosuccinates,

33 µg

 

19 ± 4

22 ± 8

37 ± 8

201 ± 8

58 ± 24

sodium salt

100 µg

 

18 ± 5

21 ± 5

44 ± 3

199 ± 6

52 ± 2

 

333 µg

 

20 ± 6

25 ± 1

46 ± 5

197 ± 13

55 ± 7

 

1000 µg

 

21 ± 4

17 ± 5

34 ± 9

195 ± 16

59 ± 4

 

2500 µg

 

16 ± 8P

16 ± 6P

31 ± 8P

208 ± 19P

65 ± 3P

 

5000 µg

 

14 ± 4P

14 ± 2P

38 ± 5P

195 ± 14P

61 ± 8P

2-AA

2.5 µg

 

347 ± 19

292 ± 10

1749 ± 240

2236 ± 355

 

2-AA

10.0 µg

 

 

 

 

 

351 ± 47

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

Precipitate

1.1     Summary of Results Experiment II

Study Name: 1491203

Study Code: Harlan CCR 1491203

Experiment: 1491203 HV2 Pre

Date Plated: 25/07/2012

Assay Conditions:

Date Counted: 01/08/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

21 ± 2

20 ± 6

29 ± 12

166 ± 17

50 ± 6

Untreated

 

 

17 ± 3

21 ± 2

34 ± 10

196 ± 24

53 ± 12

Alkenes,

33 µg

 

20 ± 6

21 ± 7

28 ± 7

177 ± 7

50 ± 10

hydroformylated

100 µg

 

21 ± 3

19 ± 1

29 ± 5

188 ± 8

52 ± 4

sulfosuccinates,

333 µg

 

16 ± 2

18 ± 2

29 ± 5

180 ± 8

55 ± 10

sodium salt

1000 µg

 

16 ± 4

16 ± 1

23 ± 4

135 ± 7

45 ± 6

 

2500 µg

 

8 ± 2P

11 ± 4P

23 ± 3P

107 ± 11P

42 ± 7P

 

5000 µg

 

5 ± 1P

9 ± 6P

18 ± 9P

82 ± 9P

44 ± 3P

NaN3

10 µg

 

1614 ± 30

 

 

1901 ± 74

 

4-NOPD

10 µg

 

 

 

755 ± 21

 

 

4-NOPD

50 µg

 

 

106 ± 20

 

 

 

MMS

3.0 µL

 

 

 

 

 

279 ± 39

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

25 ± 5

30 ± 1

46 ± 4

226 ± 6

65 ± 8

Untreated

 

 

28 ± 5

35 ± 3

48 ± 5

202 ± 25

61 ± 6

Alkenes,

33 µg

 

25 ± 1

30 ± 4

41 ± 7

204 ± 21

69 ± 14

hydroformylated

100 µg

 

28 ± 2

30 ± 2

54 ± 10

224 ± 16

62 ± 4

sulfosuccinates,

333 µg

 

25 ± 2

32 ± 4

44 ± 9

217 ± 14

66 ± 8

sodium salt

1000 µg

 

24 ± 2

27 ± 2

47 ± 3

215 ± 16

70 ± 1

 

2500 µg

 

24 ± 4P

22 ± 9P

46 ± 10P

187 ± 17P

66 ± 10P

 

5000 µg

 

15 ± 5P

21 ± 5P

36 ± 4P

157 ± 10P

58 ± 5P

2-AA

2.5 µg

 

288 ± 22

252 ± 26

1825 ± 124

1583 ± 114

 

2-AA

10.0 µg

 

 

 

 

 

222 ± 14

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item Alkenes hydroformylated, sulfosuccinates, sodium salt was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                    33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1535 at 2500 and 5000 µg/plate in the absence of metabolic activation. No toxic effects were observed in the remaining strain with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Alkenes hydroformylated, sulfosuccinates, sodium salt at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutage­nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.